Membrane phospholipids are essential for cell survival and proliferation. Cells maintain unique membrane characteristics while responding to stimuli and doubling their macromolecular components prior to division. The mechanisms that supply and control the cellular membrane phosphatidylcholine (PtdCho) content are the focus of this research. PtdCho is the major structural building block of biological membranes and is the precursor to the other two major phospholipids in cultured cells. The CTP:phosphocholine cytidylyltransferase (CCT) controls the biosynthesis of the choline headgroup and the recent discovery of a second CCT isoform (CCTbeta) reveals a new regulatory mechanism that will be investigated. CCTalpha and CCTbeta are products of different genes and differ in their post-translational modification and cellular location. The regulation of their activity by interaction with lipids will be biochemically characterized and the importance of this mechanism evaluated in vivo. The non-redundant physiological contribution of CCTbeta to PtdCho biosynthesis will be evaluated following targeted disruption of the murine CCTbeta gene. Less is known about the processes that control the supply of the diacylglycerol (DG) required for PtdCho synthesis. Our working hypothesis is that fatty acid and DG synthesis are regulated by growth factors whereas membrane PtdCho accumulation, regulated largely by CCT activity, fluctuates periodically with the cell cycle. The metabolic origin of DG after mitogenic stimulation and the fate of biosynthetic DG under conditions of restricted PtdCho synthesis will be evaluated using metabolic radiolabeling experiments. Cells also maintain a setpoint for membrane PtdCho content and experiments are proposed to characterize the cellular response to excess phospholipid production. The final step in the PtdCho biosynthetic pathway is catalyzed by the choline phosphotransferase (CPT) and cDNAs for two CPT isoforms have been isolated. CPT will be biochemically characterized following heterologous expression in insect cells and the role of the reverse reaction in controlling membrane PtdCho content will be evaluated in cultured cells. The cellular site(s) for PtdCho synthesis will be determined with specific immunological reagents and laser-scanning confocal immunofluorescent microscopy. This research will advance our understanding of the factors that influence cell membrane synthesis and trafficking and then mechanisms that control phospholipid homeostasis in growing cells.